Biosensors have been developed to detect a variety of biomolecular complexes including oligonucleotides, antibody-antigen interactions, hormone-receptor interactions, and enzyme-substrate interactions. In general, biosensors consist of two components: a highly specific recognition element and a transducer that converts the molecular recognition event into a quantifiable signal. Such signal transduction may be accomplished by many methods, including fluorescence, interferometry, and gravimetry.
Direct methods that do not require labeling of analytes with fluorescent compounds are of interest due to the relative assay simplicity and ability to study the interaction of small molecules and proteins that are not readily labeled. Direct optical methods include surface plasmon resonance (SPR), grating couplers, ellipsometry, evanescent wave devices, and reflectometry. Theoretically predicted detection limits of these detection methods have been determined and experimentally confirmed to be feasible down to diagnostically relevant concentration ranges.
Typically in real-time studies of biomolecular complexes or interactions, immobilized ligand molecules attached to the surface of a sensor-chip e.g. through a chemical binding layer are brought into contact with analyte molecules. Thereupon interaction ensues which may be monitored by one or more of the aforementioned methods of detection. As the efficacy of such studies may depend primarily on the ligand molecules, efficient immobilization of ligand molecules is often a basic and most crucial requirement. High density of ligand molecules increases the amount of analyte molecules that interact therewith and thus afford a more sensitive measurement with a lower limit of detection.
In addition to the amount of immobilized ligand, another critical aspect is the maintenance of the biological activity of the ligand upon its attachment to the surface. Conservation of high ligand activity guarantees that large number of analyte molecules would interact with the ligand, yielding a more sensitive assay. The ligand activity is strongly dependant on the biocompatibility of the binding layer, as well as on various parameters of the immobilization method and process.
Improved assay sensitivity is necessary especially in the study of interactions between macromolecules such as proteins and small molecules, whereas a result of the differences in size between the protein ligand and the small analyte molecule, the resulting signal may be relatively low. Such bioassays are of special importance in the area of drug discovery, in which measuring the binding of small target molecules to large protein-based receptors is often sought.
One of the most accepted methods for attachment of ligand molecules to a surface is based on using binding layers that contain carboxylic groups (CGs), present in the acid form and/or as a corresponding carboxylate salt. The CGs are commonly activated to form reactive esters that then react with nucleophilic groups of the ligand molecules, mainly with primary amine groups to form covalent amide bonds. The most commonly used activation solutions consists of a mixture of a water-soluble carbodiimide, most often N-ethyl-N′-(3-dimethylaminopropyl) carbodiimide (EDC), and N-hydroxysuccinimide (NHS), to form reactive NHS esters. European Patent No. 1343010 teaches an alternative, less common procedure, in which N-hydroxysulfosuccinimide (sulfo-NHS) is used instead of NHS.
Among the various types of such binding layers, a frequently used layer in commercially-available biosensors is based on carboxymethyl dextran (CMD), as taught for example in Patents Nos. WO09221976 and U.S. Pat. No. 5,436,161, and demonstrated in scientific articles such as J. Chem. Soc. Chem. Commun. 1990, 1526-1528 and Anal. Biochem. 1991, 198, 268-277. It was reported that maximum 30-40% of the CGs in this layer can be activated to NHS esters using standard activation solutions. Additionally, it was shown that the CMD layer usually binds only part of the protein amount that was electrostatically adsorbed to it. In some cases, the binding was practically insufficient for performance of an interaction assay.
From another aspect, it was demonstrated that the activated layer has net negative electrostatic charge due to the presence of non-activated CGs. It is clear that this charge remains also after common deactivation step, in which the activated groups are reacted with ethanolamine to form neutral amides. The remaining charge is undesired at the interaction assay stage, since charged analyte molecules might interact non-specifically with the layer, causing possible interruptions and distortions in the assay results, as familiar to any person skilled in the art.
More efficient binding of ligand molecules can be obtained by direct coupling, or alternatively by using chemical or biological capturing moiety, such as biotin or avidin, antibodies, disulfide for thiol coupling, etc., as taught for example in Biosensors Bioelec. 1995, 10, 813-822. Layers amenable for more efficient activation are expected to be superior also when such moieties are used, due to higher amounts of bound capturing molecules.
Furthermore, the residual charge of such improved layers can be minimized more efficiently upon deactivation process, yielding more favorable environment for the interaction assay stage.
Thus, there exists the need for improved layers which are more amenable for activation, since they have potential to bind ligand molecules more efficiently and thus increase the assay sensitivity and lower the limit of detection.